It has now become common to insert heterologous DNA into plants, including both monocots and dicots. Vectors used to carry out such insertions, or “transform” the plants, include Agrobacterium vectors and ballistic vectors. Unfortunately, when plant transformations are routinely performed, the resulting transformants frequently express the transgene at unpredictable levels or at inappropriate times. And while it is true that plant transformation has become “routine”, it is also true that plants vary widely with respect to their ability to be transformed, some plants being largely recalcitrant to transformation. For such difficult-to-transform plants therefore, it would be desirable to have higher efficiency transformation procedures and vectors, which are provided by the present invention.
The lack of predictability for expressing a genetic trait (for example, herbicide resistance) increases the cost of producing the desired plant because many “undesired” plants have to be initially screened to get the desired plant. The need to screen many transformants decreases the value of the transgenic crop and decreases confidence in the use of transgenic materials. Hence, it would be desirable to provide ways to introduce heterologous nucleic acids of interest into pre-established “target” sites within the genome of the plant to be transformed, where the DNA target site has been previously chosen to provide stable and predictable expression of the heterologous nucleic acid.
PCT Application WO99/25821 to Baszczynski et al. (assigned to Pioneer Hi-Bred) describes methods for the targeted excision of nucleotide sequences from a plant genome. P. Hooykaas describes the targeted delivery of a heterologous DNA by Agrobacterium-mediated transformation in Arabidopsis with the Cre/LOX site-specific recombination system (A. Vergunst et al., Nucleic Acids Res. 26, 2729 (1998); A. Vergunst and P. Hooykaas., Plant Molec. Biol 38, 393–406 (1998). However, high-efficiency targeted insertion of a desired nucleic acid into a plant of interest at a predetermined chromosomal site has not yet been described.
Transformation involves the insertion and integration of exogenous DNA into the genome of a cell by physical or biological means. True transformation in a precise and targeted manner, at a reasonably high efficiency, is a difficult, complex process. Accordingly, there is a continued a need in the art for such procedures.